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Molecular characterization of hepcidin in the Asian seabass (Lates calcarifer) provides insights into its role in innate immune response
Abstract
A major challenge in the aquaculture industry is the prevalence of diseases that results in large economic losses annually. Therefore, a detailed understanding of the components in the fish immune system is necessary to develop novel methods for disease management. Hepcidin is an acute phase protein that possesses antimicrobial properties and functions as a major iron regulator. The aims of this study were to generate the complete coding sequence of hepcidin in the Asian seabass (Lates calcarifer) and to characterize its expression during an immune response. The complete open reading frame of L. calcarifer hepcidin (Lcahep), which consisted of 279 nucleotides was amplified, cloned and sequenced. The phylogenetic reconstruction using Maximum Likelihood clustered Lcahep with other seawater fish and separated it from the freshwater fish. Results of the analysis also provides additional support for the hypothesis that the evolution of hepcidin is influenced by the environment in which it is found. Additionally, the multiple sequence alignment and phylogenetic analysis indicated that Lcahep is a HAMP2-type hepcidin subunit. This was further supported by expression profiles of Lcahep in various tissues from immune-challenged L. calcarifer. The rapid up-regulation of Lcahep in the liver, spleen, kidney and gill tissues suggested an important role for Lcahep in the innate immune response against pathogens. Taken together, the results of this study clarified the identity of a hepcidin subunit in L. calcarifer and indicated an important role for this gene in the early stages of infection.
... HAMP in teleost fishes is particularly diverse (Lee et al., 2012;Yang et al., 2007), with a degree of subfunctionalization as in the case of HAMP1 which is associated with iron metabolism, and HAMP2 that plays an antimicrobial role (Neves et al., 2015). Over the last decade, HAMP properties have been explored in fish to differentiate the role of HAMP as an effective antimicrobial peptide and in iron homeostasis (Álvares et al., 2016). ...
Hepcidins are cysteine-rich peptides, which participate in iron metabolism regulation, the inflammatory and antimicrobial response. This study characterizes the hepcidin-1 (HAMP1) gene, its transcript expression in different tissues, as well as its regulation in a model of brain injury in Piaractus brachypomus. Bioinformatic analysis was carried out to determine conserved domains, glycosylation sites and protein structure of HAMP1, and probability that HAMP1 corresponds to an antimicrobial peptide (AMP). Relative gene expression of the P. brachypomus HAMP1 gene was determined by qPCR from cDNA of several tissues, a brain injury model, an organophosphate sublethal toxicity model and anesthetic experiment using the 2-ΔΔCt method. HAMP1 ORF encodes for a 91 aa pre-prohepcidin conformed for a prodomain with 42 aa and mature peptide of 25 aa. Mature domain was determined as an AMP. HAMP1 transcript is expressed in all the tissues, being higher in the spleen and liver. HAMP1 mRNA level was upregulated in the brain injury group, as well as in the olfactory bulb, optic chiasm and telencephalon of red-bellied pacu brain exposed to an organophosphate. In anesthetic experiment, HAMP1 mRNA level was upregulated in the liver and gills. HAMP1 gene of P. brachypomus may be involved in the inflammatory, antimicrobial, hypoxia and stress oxidative response.
... Earlier studies found that the expression of hepcidin is upregulated during infection, inflammation, and iron overload and predominantly reduced under certain conditions like anemia and hypoxia (Ganz 2007). The development of hepcidins are assorted in teleost fishes as it is influenced by the diversity of aquatic environments in which it is found (Lee et al. 2012). It has been found that the two hepcidin peptides identified in teleost fish show a degree of sub functionalization of its functions, where hepcidin 1 (HAMP1) more implicated in iron metabolism regulation and hepcidin 2 (HAMP2) mainly performing an antimicrobial role (Neves et al. 2015). ...
Background
Antimicrobial peptides were examined as an evolutionarily preserved component of the innate immune response and identified as a vital first-line defense against a broad spectrum of pathogens in fish. The objective of this study was to investigate the molecular characterization and expression analysis of hepc1 and hepc2 in three strains of tilapia species infected naturally in Lake Manzala.
Results
The results revealed that the alignment of nucleotide sequences including cDNA and deduced amino acid sequences showed that hepcidin 1 in Sarotherodon galilaeus has four genotypes due to SNPs in codon 34Gln (CAG)/Leu (CTG) and codon 36Glu (GAA/GAG). Regarding hepcidin 2 gene, different genotypes were detected in Tilapia zillii and Sarotherodon galilaeus due to SNPs in codons 19Met (ATG)/Ile (ATT), 57Pro (CCA)/Ser (TCA), and codon 14Leu (CTT / CTC). Hepcidins 1 and 2 coding region sequences in three tilapia species deposited to GeneBank and phylogenetic analysis indicated that tilapia species are more similar to each other and closely related to Sea perch. On the other hand, the expression levels of hepc1 and hepc2 genes were over-expressed in different tilapia tissue species (hepc1 in Sarotherodon galilaeus and hepc2 in Tilapia zillii).
Conclusions
The results concluded that the hepcidin 1 and 2 genes showed constitutive expressions in most of tested tissues and have a very similar three-dimensional structure as well as mature peptides which mean that these genes are highly conserved within the species examined.
... Asian seabass, Lates calcarifer, is a potential candidate species for farming in the Asia-Pacific region, because of its fast growth rate, tolerance to wide environmental conditions and its demand in domestic and export markets 13 . A limited number of studies have been carried out on the expression and characterization of different immune-related genes of Asian seabass in recent years [14][15][16] . However, to study the expression pattern of these and other immune genes like pattern recognition receptors (PRRs) in larval developmental stages, in infected condition and in different tissues, a suitable reference gene is required to normalize the RT-PCR data. ...
Quantitative real-time PCR (qRT-PCR), used to determine the gene expression profile, is an important tool in functional genomic research, including fishes. To obtain more robust and meaningful result, the best possible normalization of the data is of utmost significance. In the present study, we have evaluated the potential of five commonly used housekeeping genes i.e., elongation factor 1-α (EF1A), β-Actin (ACTB), 18S ribosomal RNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-2-Microglobulin (B2M) in normal physiological conditions, developmental stages and in response to bacterial infection in Asian seabass, Lates calcarifer (Bloch), an important food fish cultured in the Asia-Pacific region. The expression levels of these five genes were estimated in 11 tissues of normal seabass juveniles, 14 embryonic and larval developmental stages and six tissues of Vibrio alginolyticus-challenged animals. Further, the expression stability of these genes was calculated based on three algorithms i.e. geNorm, NormFinder and BestKeeper. The results showed that although there are tissue-specific variations for each gene, ACTB and EF1A are the most stable genes across the tissues of normal animals. However, in bacteria-challenged animals, EF1A and 18S were found to be the best reference genes for data normalization. The expression of all the genes tested showed an increasing trend in developmental stages and the increase was significant at blastula stage. Among the five genes tested, EF1A and ACTB were found to be the genes with least variation and highest stability across the developmental stages. This forms the first report on validation of housekeeping genes in L. calcarifer, in the context of ontogenic development and in response to infection. © 2016, National Institute of Science Communication. All rights reserved.
The lysosomal aspartic proteinase cathepsin D is an acute phase protein involved in various physiological processes, including vitellogenesis, yolk processing and immune responses. In this study, we characterised the cathepsin D from the Asian seabass Lates calcarifer and examined its expression profile during infection. The complete coding sequence of L. calcarifer cathepsin D consists of 1191 nucleotides, encoding a 396 amino acid protein molecule that is made up of a putative signal peptide, a leader peptide and a mature peptide. Phylogenetic analyses showed that two types of cathepsin D are present in the teleost lineage i.e. cathepsin D1 and D2, whereas higher vertebrates possess only one type of cathepsin D. L. calcarifer cathepsin D was clustered together with cathepsin D1 from other teleosts. Compared to mammalian sequences, L. calcarifer cathepsin D lacks the β-hairpin loop that forms the double chain and is present as a single chain peptide with conserved aspartic active sites like other fish. Both multiple sequence alignment and phylogenetic analysis indicated that the L. calcarifer cathepsin D sequence codes for cathepsin D1 and suggested that it shares the same functions with cathepsin D from other fish. Expression profiling analysis of cathepsin D in L. calcarifer infected with Aeromonas hydrophila showed that it is up-regulated in immune-related tissues such as gills, spleen and liver, suggesting that cathepsin D plays an important role in the innate immune response of L. calcarifer against pathogens.
The availability of massive amounts of DNA sequence information has begun to revolutionize the practice of biology. As a result, current large-scale sequencing output, while impressive, is not adequate to keep pace with growing demand and, in particular, is far short of what will be required to obtain the 3-billion-base human genome sequence by the target date of 2005. To reach this goal, improved automation will be essential, and it is particularly important that human involvement in sequence data processing be significantly reduced or eliminated. Progress in this respect will require both improved accuracy of the data processing software and reliable accuracy measures to reduce the need for human involvement in error correction and make human review more efficient. Here, we describe one step toward that goal: a base-calling program for automated sequencer traces, phred, with improved accuracy. phred appears to be the first base-calling program to achieve a lower error rate than the ABI software, averaging 40%-50% fewer errors in the data sets examined independent of position in read, machine running conditions, or sequencing chemistry.
PhyML is a phylogeny software based on the maximum-likelihood principle. Early PhyML versions used a fast algorithm performing nearest neighbor interchanges to improve a reasonable starting tree topology. Since the original publication (Guindon S., Gascuel O. 2003. A simple, fast and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52:696-704), PhyML has been widely used (>2500 citations in ISI Web of Science) because of its simplicity and a fair compromise between accuracy and speed. In the meantime, research around PhyML has continued, and this article describes the new algorithms and methods implemented in the program. First, we introduce a new algorithm to search the tree space with user-defined intensity using subtree pruning and regrafting topological moves. The parsimony criterion is used here to filter out the least promising topology modifications with respect to the likelihood function. The analysis of a large collection of real nucleotide and amino acid data sets of various sizes demonstrates the good performance of this method. Second, we describe a new test to assess the support of the data for internal branches of a phylogeny. This approach extends the recently proposed approximate likelihood-ratio test and relies on a nonparametric, Shimodaira-Hasegawa-like procedure. A detailed analysis of real alignments sheds light on the links between this new approach and the more classical nonparametric bootstrap method. Overall, our tests show that the last version (3.0) of PhyML is fast, accurate, stable, and ready to use. A Web server and binary files are available from http://www.atgc-montpellier.fr/phyml/.
Despite the central role of quantitative PCR (qPCR) in the quantification of mRNA transcripts, most analyses of qPCR data
are still delegated to the software that comes with the qPCR apparatus. This is especially true for the handling of the fluorescence
baseline. This article shows that baseline estimation errors are directly reflected in the observed PCR efficiency values
and are thus propagated exponentially in the estimated starting concentrations as well as ‘fold-difference’ results. Because
of the unknown origin and kinetics of the baseline fluorescence, the fluorescence values monitored in the initial cycles of
the PCR reaction cannot be used to estimate a useful baseline value. An algorithm that estimates the baseline by reconstructing
the log-linear phase downward from the early plateau phase of the PCR reaction was developed and shown to lead to very reproducible
PCR efficiency values. PCR efficiency values were determined per sample by fitting a regression line to a subset of data points
in the log-linear phase. The variability, as well as the bias, in qPCR results was significantly reduced when the mean of
these PCR efficiencies per amplicon was used in the calculation of an estimate of the starting concentration per sample.
Strains of Aeromonas hydrophila isolated from skin infections of common freshwater fish in Bangladesh were tested for enterotoxin production, hemolysin production, and any correlation between these two activities. We also tested the resistance patterns of A. hydrophila to different drugs, especially in relation to ampicillin. The A. hydrophila strains produced an enterotoxin that was related to their beta-hemolytic activities. Production of beta-hemolysin may thus be an indicator of enterotoxicity. As 50% of the strains of A. hydrophila were found to be susceptible to 12.5 micrograms of ampicillin per ml, media containing this antibiotic may not be suitable for their isolation.
Cysteine-rich antimicrobial peptides are abundant in animal and plant tissues involved in host defense. In insects, most are
synthesized in the fat body, an organ analogous to the liver of vertebrates. From human urine, we characterized a cysteine-rich
peptide with three forms differing by amino-terminal truncation, and we named it hepcidin (Hepc) because of its origin in
the liver and its antimicrobial properties. Two predominant forms, Hepc20 and Hepc25, contained 20 and 25 amino acid residues
with all 8 cysteines connected by intramolecular disulfide bonds. Reverse translation and search of the data bases found homologous
liver cDNAs in species from fish to human and a corresponding human genomic sequence on human chromosome 19. The full cDNA
by 5′ rapid amplification of cDNA ends was 0.4 kilobase pair, in agreement with hepcidin mRNA size on Northern blots. The
liver was the predominant site of mRNA expression. The encoded prepropeptide contains 84 amino acids, but only the 20–25-amino
acid processed forms were found in urine. Hepcidins exhibited antifungal activity against Candida albicans,Aspergillus fumigatus, and Aspergillus nigerand antibacterial activity against Escherichia coli,Staphylococcus aureus, Staphylococcus epidermidis, and group B Streptococcus. Hepcidin may be a vertebrate counterpart of cysteine-rich antimicrobial peptides produced in the fat body of insects.
The significance of Aeromonas hydrophila in association with disease outbreaks in aquaculture production in the Zhejiang province of China was investigated. Bacteriological examination of moribund fish and crabs resulted in 95 bacterial isolates: 88 bacterial isolates from fish and 7 isolates from crabs. PCR and traditional biochemical methods were used for identification of A. hydrophila. Out of 69 motile aeromonads, 35 isolates were identified as A. hydrophila by biochemical tests. However, 6 of those were not identified as A. hydrophila by a species specific PCR method. Serotyping revealed 2 dominant serotypes (O9 and O97) among A. hydrophila isolates. The data presented show that approximately 42% of the motile aeromonads isolated from disease outbreaks among various fish species were A. hydrophila. It is noteworthy that A. hydrophila accounted for more than 50% of the isolated aeromonands isolated from crucian carp Carassius carassius and Wuchang bream Megalobrama amblycephala with haemorrhagic septicaemia. Although this species was the most frequently isolated organism from internal organs of diseased fish and crabs in the present study, other motile Aeromonas spp. were also found. The PCR assay was useful in preventing misidentification of A. hydrophila, which may occur when only phenotypic tests are employed.
Tissue trauma or invasion by pathogens or parasites induce changes in the quantities of several macromolecules in animal body fluids. These changes comprise one aspect of the acute phase response (APR), which in toto involves metabolic changes in several organ systems. One clear indication of the response is the increase in synthesis and secretion by the liver of several plasma proteins, with simultaneous decreases in others. These acute phase proteins (APP) function in a variety of defense-related activities such as limiting the dispersal of infectious agents, repair of tissue damage, inactivation of proteases, killing of microbes and other potential pathogens, and restoration of the healthy state. Some APP are directly harmful to microbes, while others modify targets thus marking them for cell responses. Some work alone while others contribute to cascades. Proteins that are APP in mammals, and that have been identified in both teleosts and elasmobranchs include C-reactive protein, serum amyloid P, and several components of the Complement system. Others reported in teleosts include transferrin and thrombin. Of these, only CRP has been reported to increase in acute phase plasma. In trout, a precerebellin-like protein is an APP with unknown functions. A cDNA library enriched in fragments of transcripts that were more abundant in livers from fish undergoing an APR recently yielded sequences resembling 12 additional known APP, and as many others either not known to be APP, or not similar to others yet in public databases. It appears that, as in mammals, hepatocytes are the prime source of APP in fish, and that pro-inflammatory cytokines induce transcription of their genes.
The antibacterial and antifungal peptide hepcidin (LEAP-1) is expressed in the liver. This circulating peptide has recently
been found to also act as a signaling molecule in iron metabolism. As such, it plays an important role in hereditary hemochromatosis,
a serious iron overload disease. In this study, we report the solution structures of the hepcidin-20 and -25 amino acid peptides
determined by standard two-dimensional 1H NMR spectroscopy. These small cysteine-rich peptides form a distorted β-sheet with an unusual vicinal disulfide bridge found
at the turn of the hairpin, which is probably of functional significance. Both peptides exhibit an overall amphipathic structure
with six of the eight Cys involved in maintaining interstrand connectivity. Hepcidin-25 assumes major and minor conformations
centered about the Pro residue near the N-terminal end. Further NMR diffusion studies indicate that hepcidin-20 exists as
a monomer in solution, whereas hepcidin-25 readily aggregates, a property that may contribute to the different activities
of the two peptides. The nuclear Overhauser enhancement spectroscopy spectra of the hepcidin-25 aggregates indicate an interface
for peptide interactions that again involves the first five residues from the N-terminal end.
The acute-phase response consists in a large number of behavioural, physiologic, biochemical, and nutritional changes involving many organ systems distant from the site, or sites, of inflammation. One of the most investigated, but still not well understood, characteristic of the acute phase is the up-regulation, or down- regulation, of many plasma proteins, known as the acute-phase proteins. The changes in the concentrations of these positive acute-phase proteins and negative acute-phase proteins are due to changes in their liver production. Their increase may vary from 25 percent to 1000 fold, as in the case of C-reactive protein and serum amyloid A. This review summarises the recent advances that have been acquired on the acute-phase proteins, in particular their function in pathologies such as infections or inflammatory lesions.
Quantification of mRNAs using real-time polymerase chain reaction (PCR) by monitoring the product formation with the fluorescent dye SYBR Green I is being extensively used in neurosciences, developmental biology, and medical diagnostics. Most PCR data analysis procedures assume that the PCR efficiency for the amplicon of interest is constant or even, in the case of the comparative C t method, equal to 2. The latter method already leads to a 4-fold error when the PCR efficiencies vary over just a 0.04 range. PCR efficiencies of amplicons are usually calculated from standard curves based on either known RNA inputs or on dilution series of a reference cDNA sample. In this paper we show that the first approach can lead to PCR efficiencies that vary over a 0.2 range, whereas the second approach may be off by 0.26. Therefore, we propose linear regression on the Log(fluorescence) per cycle number data as an assumption-free method to calculate starting concentrations of mRNAs and PCR efficiencies for each sample. A computer program to perform this calculation is available on request (e-mail: [email protected]
/* */; subject: LinRegPCR).
Antimicrobial peptides play a crucial role as the first line of defense against invading pathogens. Several types of antimicrobial peptides have been isolated from fish, mostly of the cationic alpha-helical variety. Here, we present the cDNA sequences of five highly disulphide-bonded hepcidin-like peptides from winter flounder, Pseudopleuronectes americanus (Walbaum) and two from Atlantic salmon, Salmo salar (L.). These hepcidin-like molecules consist of a 24 amino acid signal peptide and an acidic propiece of 38-40 amino acids in addition to the mature processed peptide of 19-27 amino acids. Exhaustive data mining of GenBank with these sequences revealed that similar peptides are encoded in the genomes of Japanese flounder, rainbow trout, hybrid striped bass and medaka, indicating that they are widespread among fish. Southern hybridization analysis suggests that closely related hepcidin-like genes are present in other flatfish species, and that they exist as a multigene family clustered on the winter flounder genome. Hepcidin variants are differentially expressed during bacterial challenge, during larval development of P. americanus and in different tissues of adult fish.
Hepcidin is a 25-amino acid peptide involved in iron homeostasis in mice and humans. It is produced in the liver from a larger precursor, and it is detectable in blood and urine. In contrast to the human genome, which contains only one copy of the gene, the mouse genome contains 2 highly similar hepcidin genes, hepc1 and hepc2, which are, however, considerably divergent at the level of the corresponding mature 25-amino acid peptide. This striking observation led us to ask whether hepc1 and hepc2 performed the same biologic activity with regard to iron metabolism in the mouse. We recently described the severe iron-deficient anemia phenotype in transgenic mice overexpressing hepc1 in the liver. Here we report that, in contrast to the hepc1-transgenic mice, none of the 7 founder hepc2-transgenic animals suffered from anemia. They all developed normally with hematologic parameters similar to the nontransgenic littermates. Hepc2 transgenic mRNA level was found to be very high for all lines compared with the level of hepc1 transgene mRNA necessary to produce severe anemia. These data provide evidence that hepc2 does not act on iron metabolism like hepc1 and give clues for the identification of amino acids important for the iron-regulatory action of the mature 25-amino acid peptide.
PerlPrimer is a cross-platform graphical user interface application for the design of primers for standard, bisulphite and
real-time PCR, and sequencing. The program incorporates highly accurate melting-temperature and primer–dimer prediction algorithms
with powerful tools such as sequence retrieval from Ensembl and the ability to BLAST search primer pairs. It aims to automate
and simplify the process of primer design.
Availability: Open-source and freely available from http://perlprimer.sourceforge.net
Hypoferremia is a common response to systemic infections or generalized inflammatory disorders. In mouse models, the development of hypoferremia during inflammation requires hepcidin, an iron regulatory peptide hormone produced in the liver, but the inflammatory signals that regulate hepcidin are largely unknown. Our studies in human liver cell cultures, mice, and human volunteers indicate that IL-6 is the necessary and sufficient cytokine for the induction of hepcidin during inflammation and that the IL-6-hepcidin axis is responsible for the hypoferremia of inflammation.
Ferroportin 1 (FPN1) is transmembrane protein involved in iron homeostasis. In the duodenum, FPN1 localizes to the basolateral surface of enterocytes where it appears to export iron out of the cell and into the portal circulation. FPN1 is also abundantly expressed in reticuloendothelial macrophages of the liver, spleen, and bone marrow, suggesting that this protein serves as an iron exporter in cells that recycle iron from senescent red blood cells. To directly test the hypothesis that FPN1 functions in the export of iron after erythrophagocytosis, FPN1 was stably expressed in J774 mouse macrophages by using retroviral transduction, and release of ⁵⁹Fe after phagocytosis of ⁵⁹Fe-labeled rat erythrocytes was measured. J774 cells overexpressing FPN1 released 70% more ⁵⁹Fe after erythrophagocytosis than control cells, consistent with a role in the recycling of iron from senescent red cells. Treatment of cells with the peptide hormone hepcidin, a systemic regulator of iron metabolism, dramatically decreased FPN1 protein levels and significantly reduced the efflux of ⁵⁹Fe after erythrophagocytosis. Subsequent fractionation of the total released ⁵⁹Fe into heme and nonheme compounds revealed that hepcidin treatment reduced the release of nonheme ⁵⁹Fe by 50% and 25% from control and FPN1-overexpressing cells, respectively, but did not diminish efflux of ⁵⁹Fe-heme. We conclude that FPN1 is directly involved in the export of iron during erythrocyte-iron recycling by macrophages.
• reticuloendothelial cell
• reticuloendothelial system
Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. The cysteine-rich AMPs such as defensin and hepcidin have been extensively studied from various organisms, but their role in disease defense in catfish is unknown. As a first step, we sequenced a hepcidin cDNA from both channel catfish and blue catfish, and characterized the channel catfish hepcidin gene. The channel catfish hepcidin gene consists of two introns and three exons that encode a peptide of 96 amino acids. The amino acid sequences and gene organization were conserved between catfish and other organisms. In contrast to its almost exclusive expression in the liver in humans, the channel catfish hepcidin gene was expressed in a wide range of tissues except brain. Its expression was detected early during embryonic and larval development, and induced after bacterial infection with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC) in a tissue-specific manner. The upregulation was observed in the spleen and head kidney, but not in the liver. The expression of hepcidin was upregulated 1--3 days after challenge, but returned to normal levels at 7 days after challenge. The expression profile of the catfish hepcidin gene during the course of bacterial infection mirrors those of inflammatory proteins such as chemokines, suggesting an important role for hepcidin during inflammatory responses.
Hepcidin is a cysteine-rich cationic antimicrobial peptide central to iron metabolism. We report a comparative analysis of the sequences, gene organization and expression of two hepcidin genes from olive flounder Paralichthys olivaceus. Both consist of two introns and three exons that encode a prepropeptide (81 amino acids for hepcidin I and 89 amino acids for hepcidin II). A TATA box and several consensus-binding motifs for transcription factors were found upstream of the transcriptional starting site. Hepcidin II was predominantly expressed in the liver and highly inducible under the effect of lipopolysaccharide (LPS), while a large amount of hepcidin I transcripts was detected in various tissues but did not appear to have a significant effect during LPS-stimulation.
The integrity of RNA molecules is of paramount importance for experiments that try to reflect the snapshot of gene expression at the moment of RNA extraction. Until recently, there has been no reliable standard for estimating the integrity of RNA samples and the ratio of 28S:18S ribosomal RNA, the common measure for this purpose, has been shown to be inconsistent. The advent of microcapillary electrophoretic RNA separation provides the basis for an automated high-throughput approach, in order to estimate the integrity of RNA samples in an unambiguous way.
A method is introduced that automatically selects features from signal measurements and constructs regression models based on a Bayesian learning technique. Feature spaces of different dimensionality are compared in the Bayesian framework, which allows selecting a final feature combination corresponding to models with high posterior probability.
This approach is applied to a large collection of electrophoretic RNA measurements recorded with an Agilent 2100 bioanalyzer to extract an algorithm that describes RNA integrity. The resulting algorithm is a user-independent, automated and reliable procedure for standardization of RNA quality control that allows the calculation of an RNA integrity number (RIN).
Our results show the importance of taking characteristics of several regions of the recorded electropherogram into account in order to get a robust and reliable prediction of RNA integrity, especially if compared to traditional methods.
Many secretory proteins and peptides are synthesized as inactive precursors that in addition to signal peptide cleavage undergo post-translational processing to become biologically active polypeptides. Precursors are usually cleaved at sites composed of single or paired basic amino acid residues by members of the subtilisin/kexin-like proprotein convertase (PC) family. In mammals, seven members have been identified, with furin being the one first discovered and best characterized. Recently, the involvement of furin in diseases ranging from Alzheimer's disease and cancer to anthrax and Ebola fever has created additional focus on proprotein processing. We have developed a method for prediction of cleavage sites for PCs based on artificial neural networks. Two different types of neural networks have been constructed: a furin-specific network based on experimental results derived from the literature, and a general PC-specific network trained on data from the Swiss-Prot protein database. The method predicts cleavage sites in independent sequences with a sensitivity of 95% for the furin neural network and 62% for the general PC network. The ProP method is made publicly available at http://www.cbs.dtu.dk/services/ProP.
Determining the subcellular localization of a protein is an important first step toward understanding its function. Here, we describe the properties of three well-known N-terminal sequence motifs directing proteins to the secretory pathway, mitochondria and chloroplasts, and sketch a brief history of methods to predict subcellular localization based on these sorting signals and other sequence properties. We then outline how to use a number of internet-accessible tools to arrive at a reliable subcellular localization prediction for eukaryotic and prokaryotic proteins. In particular, we provide detailed step-by-step instructions for the coupled use of the amino-acid sequence-based predictors TargetP, SignalP, ChloroP and TMHMM, which are all hosted at the Center for Biological Sequence Analysis, Technical University of Denmark. In addition, we describe and provide web references to other useful subcellular localization predictors. Finally, we discuss predictive performance measures in general and the performance of TargetP and SignalP in particular.
The accuracy and scalability of multiple sequence alignment (MSA) of DNAs and proteins have long been and are still important
issues in bioinformatics. To rapidly construct a reasonable MSA, we developed the initial version of the MAFFT program in
2002. MSA software is now facing greater challenges in both scalability and accuracy than those of 5 years ago. As increasing
amounts of sequence data are being generated by large-scale sequencing projects, scalability is now critical in many situations.
The requirement of accuracy has also entered a new stage since the discovery of functional noncoding RNAs (ncRNAs); the secondary
structure should be considered for constructing a high-quality alignment of distantly related ncRNAs. To deal with these problems,
in 2007, we updated MAFFT to Version 6 with two new techniques: the PartTree algorithm and the Four-way consistency objective
function. The former improved the scalability of progressive alignment and the latter improved the accuracy of ncRNA alignment.
We review these and other techniques that MAFFT uses and suggest possible future directions of MSA software as a basis of
comparative analyses. MAFFT is available at http://align.bmr.kyushu-u.ac.jp/mafft/software/.
Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT-PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.
In vertebrates, the growth and maturation of the ovarian follicle is dependent on the appropriate dynamics of sex steroid secretion, which is dictated by gene expression of the steroidogenic enzymes. The molecular aspects of steroid regulation are poorly understood in fishes, so as a first step we determined the pattern of expression of four key steroidogenic genes throughout the ovarian cycle in an annually spawning teleost, the channel catfish (Ictalurus punctatus). The abundance of transcripts encoding 3β-hydroxysteroid dehydrogenase (3β-HSD) and cholesterol side chain cleavage (P450scc), 17α-hydroxylase/lyase (P450c17), and aromatase (P450arom) were determined by rtqRT-PCR or ribonuclease protection assay and correlated to ovarian growth and plasma titers of estradiol (E2) and testosterone (T) in two populations of catfish. Elevations in transcript abundance for P450c17, P450scc, and P450arom were observed at the onset of ovarian recrudescence and during early vitellogenic growth of the oocytes; however, all three decreased precipitously with the completion of vitellogenesis. Changes in the expression of these genes strongly suggest a direct correlation to E2 and T titers. Alternatively 3β-HSD transcript abundance was relatively stable throughout the year. This study suggests that the genes encoding the three steroidogenic cytochrome P450s have a similar regulatory mechanism.
Real-time reverse transcription followed by polymerase chain reaction (RT–PCR) is the most suitable method for the detection
and quantification of mRNA. It offers high sensitivity, good reproducibility and a wide quantification range. Today, relative
expression is increasingly used, where the expression of a target gene is standardised by a non-regulated reference gene.
Several mathematical algorithms have been developed to compute an expression ratio, based on real-time PCR efficiency and
the crossing point deviation of an unknown sample versus a control. But all published equations and available models for the
calculation of relative expression ratio allow only for the determination of a single transcription difference between one
control and one sample. Therefore a new software tool was established, named REST© (relative expression software tool), which
compares two groups, with up to 16 data points in a sample and 16 in a control group, for reference and up to four target
genes. The mathematical model used is based on the PCR efficiencies and the mean crossing point deviation between the sample
and control group. Subsequently, the expression ratio results of the four investigated transcripts are tested for significance
by a randomisation test. Herein, development and application of REST© is explained and the usefulness of relative expression
in real-time PCR using REST© is discussed. The latest software version of REST© and examples for the correct use can be downloaded
at http://www.wzw.tum.de/gene-quantification/.
Low environmental temperatures are immunosuppressive for ectothermic vertebrates, such as teleosts. In particular the available data support the notion that, at least in channel catfish, virgin T cells, rather than memory T cells, B cells or accessory cells are particularly susceptible to the inhibitory influences of lower temperatures on adaptive immune responses. The probable mechanisms involved in such suppression in teleosts are reviewed, and considerations are offered regarding cause and effect relationships between such immunosuppression and the development of infectious diseases in fish.
Hepcidin, an antimicrobial and iron-regulating peptide, is a key molecule of the innate immune system of bony fish. In this study, four isoforms of hepcidin genes were characterized from a marine Perciform fish, rockbream (Oplegnathus fasciatus), and the transcriptional modulations of these isoforms in response to different biological stimulations were also examined. All rockbream hepcidin isoform genes exhibited a tripartite structure and their promoter regions displayed typical binding motifs for the transcription factors including C/EBP, HNF, AP, NF-kβ, GATA, USF and/or STAT. Hepcidin transcripts in juvenile or fingerling tissues were dramatically induced during experimental challenges with various bacterial species, iron overload and rockbream iridovirus infection. The transcription of hepcidins was regulated in an isoform- and tissue-specific fashion. In addition, we identified for the first time that partially processed hepcidin transcripts were significantly elevated during bacterial infection and iron overload. Results from this study provide a good basis to better understand the isoform-specific role of hepcidin in the fish innate immune system.
Sepsis is a global problem which is exacerbated by increasing bacterial resistance to antibiotics. However, mechanisms of natural resistance can be extremely effective, and need to be exploited, but the availability of iron is critical for controlling bacterial growth. The diagnosis of sepsis and possible strategies for limiting iron availability are discussed.
Hepcidin antimicrobial peptides (HAMPs) are key molecules of the innate immune system against bacterial infections and in iron metabolism. In this study we report the molecular cloning and genomic characterization of four HAMP genes (referred to as HAMP1, HAMP2, HAMP3 and HAMP4) in the redbanded seabream (Pagrus auriga). All these genes possessed the eight characteristic cysteine residues involved in protein folding. No canonical sequence for convertase-mediated processing of the HAMP3 propeptide was identified. At the genomic level, all four HAMP genes consisted of two introns and three exons. Phylogenetic analysis revealed that HAMPs could group in two main clusters with HAMP2, HAMP3 and HAMP4 belonging to the more complex and diversified HAMP2-like group of acanthopterygians. Quantitation of mRNA levels in adult tissues showed that HAMP1 was ubiquitously expressed, HAMP2 mainly in kidney, spleen and intestine, whereas HAMP3 and HAMP4 in liver. During development, HAMP2 and HAMP3 were expressed at a high level in embryos. Moreover, the expression levels of the four HAMP genes increased between 5 and 15 days after hatching when larvae started external feeding. Induction experiments with lipopolysaccharide revealed significant changes in gene expression of the four HAMP genes in kidney, liver and spleen. However, expression profiles differed in magnitude and time course response. HAMP1 mRNAs increased rapidly in kidney at 1 h p.i. whereas HAMP2 did later at 24 h. Moreover, HAMP4 transcripts increased more than 5000-fold in liver whereas HAMP2 mRNAs dropped significantly in spleen at 3 h p.i. All these data suggest that HAMPs are involved in the response against bacterial infections although additional functions in iron regulation and embryogenesis in fish should be considered.
Mammalian iron homeostasis is regulated by the interaction of the liver-produced peptide hepcidin and its receptor, the iron transporter ferroportin. Hepcidin binds to ferroportin resulting in degradation of ferroportin and decreased cellular iron export. We identify the hepcidin-binding domain (HBD) on ferroportin and show that a synthetic 19 amino acid peptide corresponding to the HBD recapitulates the characteristics and specificity of hepcidin binding to cell-surface ferroportin. The binding of mammalian hepcidin to ferroportin or the HBD shows an unusual temperature dependency with an increased rate of dissociation at temperatures below 15 degrees C. The increased rate of dissociation is due to temperature- dependent changes in hepcidin structure. In contrast, hepcidin from poikilothermic vertebrates, such as fish or frogs, binds the HBD in a temperature-independent fashion. The affinity of hepcidin for the HBD permits a rapid, sensitive assay of hepcidin from all species and yields insights into the evolution of hepcidin.
An efficient means for generating mutation data matrices from large numbers of protein sequences is presented here. By means of an approximate peptide-based sequence comparison algorithm, the set sequences are clustered at the 85% identity level. The closest relating pairs of sequences are aligned, and observed amino acid exchanges tallied in a matrix. The raw mutation frequency matrix is processed in a similar way to that described by Dayhoff et al. (1978), and so the resulting matrices may be easily used in current sequence analysis applications, in place of the standard mutation data matrices, which have not been updated for 13 years. The method is fast enough to process the entire SWISS-PROT databank in 20 h on a Sun SPARCstation 1, and is fast enough to generate a matrix from a specific family or class of proteins in minutes. Differences observed between our 250 PAM mutation data matrix and the matrix calculated by Dayhoff et al. are briefly discussed.
Following injection challenge of rainbow trout with the Gram-positive pathogen Renibacterium salmoninarum, serum nitrate levels increased indicative of NO production. The timing and amount of nitrate produced varied with the virulence of the bacterial strain used, with the highest levels seen in fish challenged with the most virulent (autoaggregating) strain. Immunization with a killed R. salmoninarum preparation in Freund's incomplete adjuvant significantly increased nitrate levels after challenge. Inducible nitric oxide synthase (iNOS) transcript expression was detectable in rainbow trout tissues after injection challenge with R. salmoninarum, and its induction in the gills was both quick (between 3 and 6 hr) and relatively prolonged (lasting several days). iNOS expression in the kidney was also seen at a later stage (24 hr) but appeared to switch off relatively rapidly. Bath challenge with R. salmoninarum also induced iNOS expression in gill, and a variable expression in the gut and kidney also occurred. These results highlight the importance of the gills, not only as a point of entry of pathogens but also as a tissue capable of mounting an immune response.
It is becoming clear that the cationic antimicrobial peptides are an important component of the innate defenses of all species of life. Such peptides can be constitutively expressed or induced by bacteria or their products. The best peptides have good activities vs. a broad range of bacterial strains, including antibiotic-resistant isolates. They kill very rapidly, do not easily select resistant mutants, are synergistic with conventional antibiotics, other peptides, and lysozyme, and are able to kill bacteria in animal models. It is known that bacterial infections, especially when treated with antibiotics, can lead to the release of bacterial products such as lipopolysaccharide (LPS) and lipoteichoic acid, resulting in potentially lethal sepsis. In contrast to antibiotics, the peptides actually prevent cytokine induction by bacterial products in tissue culture and human blood, and they block the onset of sepsis in mouse models of endotoxemia. Consistent with this, transcriptional gene array experiments using a macrophage cell line demonstrated that a model peptide, CEMA, blocks the expression of many genes whose transcription was induced by LPS. The peptides do this in part by blocking LPS interaction with the serum protein LBP. In addition, CEMA itself has a direct effect on macrophage gene expression. Because cationic antimicrobial peptides are induced by LPS and are able to dampen the septic response of animal cells to LPS, we propose that, in addition to their role in direct and lysozyme-assisted killing of microbes, they have a role in feedback regulation of cytokine responses. We are currently developing variant peptides as therapeutics against antibiotic-resistant infections.
We report the isolation and characterization of a novel human peptide with antimicrobial activity, termed LEAP-1 (liver-expressed antimicrobial peptide). Using a mass spectrometric assay detecting cysteine-rich peptides, a 25-residue peptide containing four disulfide bonds was identified in human blood ultrafiltrate. LEAP-1 expression was predominantly detected in the liver, and, to a much lower extent, in the heart. In radial diffusion assays, Gram-positive Bacillus megaterium, Bacillus subtilis, Micrococcus luteus, Staphylococcus carnosus, and Gram-negative Neisseria cinerea as well as the yeast Saccharomyces cerevisiae dose-dependently exhibited sensitivity upon treatment with synthetic LEAP-1. The discovery of LEAP-1 extends the known families of mammalian peptides with antimicrobial activity by its novel disulfide motif and distinct expression pattern.
In vertebrates, the growth and maturation of the ovarian follicle is dependent on the appropriate dynamics of sex steroid secretion, which is dictated by gene expression of the steroidogenic enzymes. The molecular aspects of steroid regulation are poorly understood in fishes, so as a first step we determined the pattern of expression of four key steroidogenic genes throughout the ovarian cycle in an annually spawning teleost, the channel catfish (Ictalurus punctatus). The abundance of transcripts encoding 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and cholesterol side chain cleavage (P450(scc)), 17 alpha-hydroxylase/lyase (P450(c17)), and aromatase (P450(arom)) were determined by rtqRT-PCR or ribonuclease protection assay and correlated to ovarian growth and plasma titers of estradiol (E(2)) and testosterone (T) in two populations of catfish. Elevations in transcript abundance for P450(c17), P450(scc), and P450(arom) were observed at the onset of ovarian recrudescence and during early vitellogenic growth of the oocytes; however, all three decreased precipitously with the completion of vitellogenesis. Changes in the expression of these genes strongly suggest a direct correlation to E(2) and T titers. Alternatively 3 beta-HSD transcript abundance was relatively stable throughout the year. This study suggests that the genes encoding the three steroidogenic cytochrome P450s have a similar regulatory mechanism.
Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way
to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform,
provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification
of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters
into the particular topics of the relative quantification in real-time RT–PCR of a target gene transcript in comparison to
a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated
only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model
needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA
integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler
PCR using the established mathematical model.
That the plasma concentration of certain divalent cations change during an inflammatory insult provides a major host defense response in vertebrate animals. This study was designed to investigate the involvement of iron sequestration in invertebrate immune responses. A ferritin molecule was cloned from an echinoderm coelomocyte cDNA library. The amino acid sequence showed sequence homology with vertebrate ferritin. The cDNA contained a conserved iron responsive element sequence. Studies showed that stimulated coelomocytes released iron into in vitro culture supernatants. The amount of iron in the supernatants decreased over time when the amebocytes were stimulated with LPS or PMA. Coelomocytes increased expression of ferritin mRNA after stimulation. In vertebrates, cytokines can cause changes in iron levels in macrophages. Similarly, echinoderm macrokines produced decreases in iron levels in coelomocyte supernatant fluids. These results suggest that echinoderm ferritin is an acute phase protein and suggest that sequestration of iron is an ancient host defense response in animals.
Real-time reverse transcription followed by polymerase chain reaction (RT-PCR) is the most suitable method for the detection and quantification of mRNA. It offers high sensitivity, good reproducibility and a wide quantification range. Today, relative expression is increasingly used, where the expression of a target gene is standardised by a non-regulated reference gene. Several mathematical algorithms have been developed to compute an expression ratio, based on real-time PCR efficiency and the crossing point deviation of an unknown sample versus a control. But all published equations and available models for the calculation of relative expression ratio allow only for the determination of a single transcription difference between one control and one sample. Therefore a new software tool was established, named REST (relative expression software tool), which compares two groups, with up to 16 data points in a sample and 16 in a control group, for reference and up to four target genes. The mathematical model used is based on the PCR efficiencies and the mean crossing point deviation between the sample and control group. Subsequently, the expression ratio results of the four investigated transcripts are tested for significance by a randomisation test. Herein, development and application of REST is explained and the usefulness of relative expression in real-time PCR using REST is discussed. The latest software version of REST and examples for the correct use can be downloaded at http://www.wzw.tum.de/gene-quantification/.
We report the isolation of a novel antimicrobial peptide, bass hepcidin, from the gill of hybrid striped bass, white bass (Morone chrysops) x striped bass (M. saxatilis). After the intraperitoneal injection of Micrococcus luteus and Escherichia coli, the peptide was purified from HPLC fractions with antimicrobial activity against Escherichia coli. Sequencing by Edman degradation revealed a 21-residue peptide (GCRFCCNCCPNMSGCGVCCRF) with eight putative cysteines. Molecular mass measurements of the native peptide and the reduced and alkylated peptide confirmed the sequence with four intramolecular disulfide bridges. Peptide sequence homology to human hepcidin and other predicted hepcidins, indicated that the peptide is a new member of the hepcidin family. Nucleotide sequences for cDNA and genomic DNA were determined for white bass. A predicted prepropeptide (85 amino acids) consists of three domains: a signal peptide (24 amino acids), prodomain (40 amino acids) and a mature peptide (21 amino acids). The gene has two introns and three exons. A TATA box and several consensus-binding motifs for transcription factors including C/EBP, nuclear factor-kappaB, and hepatocyte nuclear factor were found in the region upstream of the transcriptional start site. In white bass liver, hepcidin gene expression was induced 4500-fold following challenge with the fish pathogen, Streptococcus iniae, while expression levels remained low in all other tissues tested. A novel antimicrobial peptide from the gill, bass hepcidin, is predominantly expressed in the liver and highly inducible by bacterial exposure.
Hepcidin is an antimicrobial peptide thought to be involved in the regulation of intestinal iron absorption. To further investigate its role in this process, we examined hepatic and duodenal gene expression in rats after the switch from a control diet to an iron-deficient diet.
Adult rats on an iron-replete diet were switched to an iron-deficient diet and the expression of iron homeostasis molecules in duodenal and liver tissue was studied over 14 days. Intestinal iron absorption was determined at these same time-points by measuring the retention of an oral dose of (59)Fe.
Iron absorption increased 2.7-fold within 6 days of switching to an iron-deficient diet and was accompanied by an increase in the duodenal expression of Dcytb, divalent metal transporter 1, and Ireg1. These changes precisely correlated with decreases in hepatic hepcidin expression and transferrin saturation. No change in iron stores or hematologic parameters was detected.
This study showed a close relationship between the expression of hepcidin, duodenal iron transporters, and iron absorption. Both hepcidin expression and iron absorption can be regulated before iron stores and erythropoiesis are affected, and transferrin saturation may signal such changes.
Motile aeromonads isolated from the intestines of farm-raised freshwater fish such as Catla catla, Labeo rohita and Ctenopharyngodon idella have been characterized to species level. Morphological and physiological grouping revealed 61% Aeromonas hydrophila, 30% Aeromonas caviae, 7% Aeromonas sobria and 2% which remained unidentified. Hemolytic activity was detected mostly in A. hydrophila, while only half of the A. sobria and A. caviae showed this activity. Antibiotic resistance patterns of the strains revealed that they had acquired a relatively higher resistance to oxytetracycline, amoxycillin, ampicillin, novobiocin and polymixin-B, implicating possible use of these antibiotics in the aquaculture systems.
A cDNA encoding hepcidin was isolated from a library of cDNA from spleen of red sea bream (Chrysophrys major) by expressed sequence tag analysis. The expression of the hepcidin mRNA in various tissues was examined. Challenge of red
sea bream with Escherichia coli DH5α elevated hepcidin mRNA levels in spleen, gill, liver, and intestine.
Hepcidin is a liver-made peptide proposed to be a central regulator of intestinal iron absorption and iron recycling by macrophages. In animal models, hepcidin is induced by inflammation and iron loading, but its regulation in humans has not been studied. We report that urinary excretion of hepcidin was greatly increased in patients with iron overload, infections, or inflammatory diseases. Hepcidin excretion correlated well with serum ferritin levels, which are regulated by similar pathologic stimuli. In vitro iron loading of primary human hepatocytes, however, unexpectedly down-regulated hepcidin mRNA, suggesting that in vivo regulation of hepcidin expression by iron stores involves complex indirect effects. Hepcidin mRNA was dramatically induced by interleukin-6 (IL-6) in vitro, but not by IL-1 or tumor necrosis factor alpha (TNF-alpha), demonstrating that human hepcidin is a type II acute-phase reactant. The linkage of hepcidin induction to inflammation in humans supports its proposed role as a key mediator of anemia of inflammation.
The cysteine-rich peptide hepcidin is known to be an antimicrobial peptide and iron transport regulator that has been found in both fish and mammals. Recently, we found two different types (designated Hep-JF1 and Hep-JF2) of hepcidin cDNA in the Japanese flounder, Paralichthys olivaceus, by expressed sequence tag analysis. The identity of amino acid sequences between Hep-JF1 and Hep-JF2 was 51%. The Hep-JF1 and Hep-JF2 genes both consist of three exons and two introns, and both exist as single copies in the genome. The predicted mature regions of Hep-JF1 and Hep-JF2 have six and eight Cys residues, respectively. The first Cys residue of Hep-JF1 was deleted and the second was replaced with Gly. The number and positions of Cys residues in Hep-JF2 are the same as they are in human Hep. Hep-JF1 is specifically expressed in liver while the expression of Hep-JF2 was detected from gill, liver, heart, kidney, peripheral blood leucocytes, spleen and stomach. Gene expression of Hep-JF1 in liver decreased during experimental iron (iron-dextran) overload. Expression of Hep-JF1 in liver was decreased by injecting fish with iron-dextran and increased by injecting lipopolysaccharide. Iron overload did not significantly affect expression of Hep-JF2 in liver but it did increase expression of Hep-JF2 in kidney. Lipopolysaccharide injection increased expression of Hep-JF2 in both liver and kidney. In liver, some cells expressed both Hep-JF1 and Hep-JF2 while some other cells expressed just one of them. Synthesized Hep-JF2 peptide showed antimicrobial activity, while synthesized Hep-JF1 peptide did not against several bacteria including fish-pathogenic bacteria used in this study.
Hepcidin is a cationic amphipathic peptide made in the liver, released into plasma and excreted in urine. Hepcidin is the homeostatic regulator of intestinal iron absorption, iron recycling by macrophages, and iron mobilization from hepatic stores, but it is also markedly induced during infections and inflammation. Under the influence of hepcidin, macrophages, hepatocytes, and enterocytes retain iron that would otherwise be released into plasma. Hepcidin acts by inhibiting the efflux of iron through ferroportin, the sole known iron exporter that is expressed in the small intestine, and in hepatocytes and macrophages. As befits an iron-regulatory hormone, hepcidin synthesis is increased by iron loading, and decreased by anemia and hypoxia. Hepcidin is also rapidly induced by cytokines, including IL-6. The resulting decrease in plasma iron levels eventually limits iron availability to erythropoiesis and contributes to the anemia associated with infection and inflammation. The decrease in extracellular iron concentrations due to hepcidin probably limits iron availability to invading microorganisms, thus contributing to host defense.
Hepcidins are antimicrobial peptides that play important roles in resisting pathogenic infection. Through hybridization of a phage library, the cDNA sequences of three hepcidin-like antimicrobial peptides (named TH1-5, TH2-2, and TH2-3) in tilapia, Oreochromis mossambicus, were determined. The complete hepcidin cDNA sequences of TH1-5, TH2-2, and TH2-3 were respectively composed of 478, 533, and 583 bases, and contained a translated region of 88, 86, and 91 amino acids. An evolutionary assay of the three deduced amino acid sequences, which share eight cysteines at identical conserved positions, showed that tilapia TH2-3 is similar to Japanese flounder (Paralichthys olivaceus) JF2, tilapia TH2-2 is similar to Japanese flounder JF1, and tilapia TH1-5 is similar to seabream (Chrysophrys major) hepcidin. The predicted molecular weights of TH1-5, TH2-2, and TH2-3 are 9.5, 9.4, and 9.8 kDa, respectively. The predicted signal peptide cleavage sites in TH1-5 is between codons 24 and 25, in TH2-2, it is between codons 22 and 23, and in TH2-3, it is between codons 24 and 25. The structural models of tilapia hepcidins, constructed using the crystal structures of bass (Morone chrysopsx M. saxatilis) hepcidin as a respective template, showed that the positional cysteine residues form disulfide bonds with tilapia hepcidin, and the cysteines likely form disulfide bonds with the bass hepcidin cysteine. The tissue-specific, lipopolysaccharide (LPS) stimulation-specific, and polyinosinic-polycytidylic acid (poly I:poly C) stimulation-specific expressions of tilapia hepcidin mRNA were determined by a comparative reverse-transcription polymerase chain reaction. Results of the tissues distribution analysis revealed high expression levels of hepcidin messenger RNA (mRNA) in the liver and head kidneys for TH1-5. TH2-3 had high mRNA expression after LPS challenge in comparison to TH2-2 and TH1-5 in fish injected with 10mug/ml LPS. TH1-5 had high mRNA expression after poly I:poly C challenge in comparison to TH2-2 and TH2-3. Immunohistochemical analysis with the polyclonal antiserum of tilapia hepcidin TH1-5 (using a rabbit polyclonal antibody) showed that the peptide was localized in the spleen and head kidneys. Synthesized TH1-5 and TH2-3 peptides showed antimicrobial activity against several bacteria in this study, while the synthesized TH 2-2 peptide did not.
Hepcidin is an antimicrobial peptide and putative iron regulatory hormone previously described in mice and humans. Dozens of fish hepcidins have been isolated and characterized so far. Here we present seven hepcidin-like cDNA sequences named AS-hepc1-7, amplified from the normal commercially cultured fish (black porgy) by RACE-PCR. Sequence analysis reveals that these seven potential hepcidin peptides have highly conserved sequences with other known hepcidins, but they are different from each other in constitution and characteristics of predicted mature amino acids. Based on the study, it is deduced that AS-hepc1-7 represent different variants of a family of hepcidin genes in black porgy. To understand the organization of these hepcidin-like genes, we sequenced AS-hepc2 DNA, AS-hepc3 DNA, AS-hepc4 DNA, AS-hepc7 DNA and AS-hepc2 upstream region; and all of the four genomic DNAs consisted of two introns and three exons, the same organization as other reported hepcidins. The tissue-specific gene expression of hepcidins in normal black porgy was evaluated using RT-PCR and dot blot approaches. RT-PCR showed that transcripts of hepcidin-like mRNAs were present in each tested tissue of normal juvenile black porgy, including liver, spleen, kidney, heart, brain, stomach, intestine, gill, skin and blood, but abundant hepcidin-like mRNA transcripts were only detected in the liver, kidney, spleen, intestine and stomach by dot blot assay. In addition, using dot blot and Northern blot approach, a significant increase of hepcidin mRNA transcription was observed in the liver within 48 h after immersion in a suspension of live bacteria, which suggested that the expression pattern of hepcidin-like genes in black porgy might be different in the liver from the other tissues as previously reported in several hepcidin studies.
Hepcidin is a small cysteine-rich peptide that plays an important role in antimicrobial activity and in maintaining iron homeostasis in vertebrates. Here we report on the underlying mechanism that maintains high sequence diversities among the hepcidin-like variants of perciform and pleuronectiform fishes. In contrast to mammals, maximum likelihood-based codon substitution analyses revealed that positive Darwinian selection (nonsynonymous to synonymous substitution, omega > 1) is the likely cause of accelerated rate of amino acid substitutions in the hepcidin mature peptide region of these fishes. Comparison of models incorporating positive selection (omega > 1) at certain sites with models not incorporating positive selection (omega < 1) failed to reject (p = 0) the evidence of positive selection among the codon sites of percifom and pleuronectiform hepcidin. The adaptive evolution of this peptide in perciform and pleuronectiform fishes might be directed by pathogens when the host is exposed to new habitats/environments.
Antimicrobial peptides (AMPs) include a diverse group of gene-encoded molecules that play a role in innate defense in many organisms. Evolutionary analyses of the AMP genes can be challenging because of gene duplication and diversification. Recently discovered, hepcidins are small, cysteine-rich antimicrobial peptides that also function as hormonal regulators of iron homeostasis. In this paper we investigated the organization, expression and molecular evolution of hepcidin. From searches of the literature and public genomic databases we collected 68 different hepcidin gene products from 51 different species, all among the vertebrates. Although some species have multiple hepcidin homologues, we suggest that each contains only one copy that functions as an iron regulator. Despite the recent report of hepcidin sequences in the pigeon (Fu, Y.M., Li, S.P., Wu, Y.F., Chang, Y.Z., 2007. Identification and expression analysis of hepcidin-like cDNAs from pigeon (Columba livia). Mol. Cell. Biochem. 305, 191-197.), searches of the chicken genomic, EST, and HTGS databases did not reveal any evidence of the presence of this gene in birds. This, along with the absence of reported avian transferrin receptor 2 and hemojuvelin sequences, suggests that iron homeostasis in birds may be regulated by an alternative mechanism.